Establishment Of HIV-1 Pseudovirus Screening System And Evaluation Of The Anti-HIV-1 Activity Of DY Series Compounds

Objective To establish and optimize the VSVG/HIV-1NL4-3 Luc pseudovirus model for anti-HIV drugs screening, and evaluation of the anti-HIV-1 activity of compounds.Methods We investigated the infectivity of VSVG/HIV-1NL4-3 Luc in 4 different cell lines according to the method of the luciferase activity analysis system of Promega company. We used 3 different experimental settings to detect the activities of approved anti-HIV drugs to confirm the feasibility and effectiveness of the system. Then, we screened some potential compounds for their anti-HIV activities, and compared their antiviral activities against the pseudovirus with HIV-1ⅢB. Finally, we choose some better compounds to detect their cytotoxicity by MTT assay and to evaluate their anti-HIV-1 activity by ELIS A on diffenert cells or different viruses.Results CRFK is the most sensitive cell to VSVG/HIV-1NL4-3 Luc, and we found a clear dose-effect relationship between the report gene expression level and the virus quantity. Comparing the EC50 of different positive inhibitors against VSVG/HIV-1NL4-3 Luc on 3 kinds of experimental conditions, the scheme 3 is the best. Next, we use the system to screen compounds, the EC50s against pseudovirus were similar to those in HIV-1ⅢB.Then, We obtained some good results to further study of some compounds:DY1108, DY1118, DY1203 and DY1204 showed small cytotoxicity in vitro on C8166,MT-4,H9 and PBMC, CC50s are greater than 200μg/ml. DY1119, DY1208 and DY1209 had broad anti-HIV-1 activity and showed strong inhibition effect on different viruses infection(include laboratory strain,drug-resistant strains and clinical isolated strains), EC50s are nanogram level. But NNRTI-resistant strain(HIV-1A17) showed varying degree resistance to them. The rest compounds also had good inhibition effect on different viruses.Lastly, we tested the inhibitory activity of compounds against the recombinant reverse transcriptase. The results showed that DY1119, DY1208 and DY1209 could inhibit the RT-associated RNA-dependent DNA polymerase activity effectively, EC50s are 0.496μg/ml、 0.038μg/ml、0.060μg/ml, and the rests had weak inhibition.Conclusion We have successfully developed an optimized VSVG/HIV-1NL4-3 Luc anti-HIV drug screening system. The platform can be used for screening NNRTIs,NRTIs and INIs, but donnot apply to PIs and entry inhibitors screening. This model can be used large-scale drug screening and evaluation anti-HIV-1 activity in BSL-2. The study of the anti-HIV-1 activity of compounds indicated that DY1119, DY1208 and DY1209 showed strong anti-HIV-1 activity in vitro on different viruses, we hope we can give some antiviral activity reference for HIV-1 drug research and promote the development of HIV-1 drugs.